Fervenulin and its production



Feb. 20, 1962 0. DE BOER ET AL 3,022,220

FERVENULIN AND ITS PRODUCTION Filed March 25, 1959 2 Sheets-Sheet 1 HONVLLIWSNVHJ. .LNHOUBd WAVE FIGURE I. INFRARED ABSORPTION EPECTRUM FERVENULIN NUMBERS IN CM" 2000 I500 I300 8 2 CLARENCE DEBOER EIONVLJJWSNVHL iNEIOHHd THOMAS E. EBLE CHARLES M. LARGE INVENTORS BY EUGENE O. RETTER GEORGE T. JOHANNESEN ATTORNEYS Feb. 20, .1962 c. DE BOER ETAL FERVENULIN AND ITS PRODUCTION Filed March 25, 1959 2 Sheets-Sheet 2 omm z 1825MB; Nmm mom m wmm kllAlldHOSGV CLARENCE DEBOER THOMAS E. EBLE CHARLES M. LARGE IN V EN TORS BYEUGENE O. RETTER GEORGE T. JOHANNESEN A TTORNE YS 3,622,220 FERVENULIN AND ITS PRODUCTION 3,022,220 Patented Feb. 20, 1962 TABLE I Assimilation of carbon compounds in synthetic medium Clarence De Boer, Kalamazoo Township, Kalamazoo Compound gfemns County, and Thomas E. Eble and Charles M. Large, 5 Kalamazoo, Mich, assignors t9 The Uin Company, Control Kalamazoo, Mich, a corporation of Delaware 1. d-Xy1ose Filed Mar. 25, 1959, Ser. No. 801,876 i ggg E3 This invention relates to a novel composition of mat- 10 6. d-Glucose. f More 7. d-l\1anno ter and to the process for the production thereo 8 particularly this invention relates to a new compound, $1 5 fervenulin (compound a-ll) and to the process for the n CeuOm-n production thereof. gggq i I Fervenulin is obtained as an elaboration product of a m" novel species of Streptomyces and has the property of i2: gig g i (i) adversely affecting the growth of various organisms, par- 17. Dnlcifnl ticularly protozoa, bacteria, and fungi. lg: g ggimg As the essential ingredient of a composition, 1t 15 use- 20. Inositol.

ful to prevent or reduce the number of organisms or the I possibility of transmission of pathogenic organisms. For 23. Cresol example, wash solutions containing fervenulin are useful g: t;

for general sanitation purposes, for example: washing of 26. Na tart-rate hands, cleaning of equipment, floors or furnishings of 3g: g2 gig g gff 1 contaminated rooms or laboratories. Its use is indi- 29. Na. citrate cated as an additive for tuberculosis room and instru- Na succmatement, Stenhzanon. for use m biological medla as an 11,1- Key: +p0sitive assimilation; -negative assimilation;

dustnal preservative, for example, as a bacteriostatic negative assimilation, but only slight growth; rinse for laundered clothes and for impregnating to make posltwe asslmflatmn out only shghf glowth' bacteriostatic Papers and fabrics, and also in teaching 30 Other cultural and morphological characteristics of research itrep tomycesl' feriigirg as shown by tl)1e Diialtn Ektaciirnorile A viable culture of the new species, identified as Posmve co or .transparencle s tee mque n S streptomyces fervens, has been deposited with the of the New York Academy of Sc1ence, vol. 60, Art. l,

Fermentation Division, Northern Utilization Research Pages 1524 October 1954 are noted In Table I Branch, U.S. Department of Agriculture, Peoria, Illinois, TABLE II and has been added to its permanent collection as Character 15176-9 f p yq fervens 2755 Ektachrome Technique Streptomyces fervens was isolated from a soil sample Ob taken at Van Nuys, California. It is particularly char- 40 Medium serva acterized by pink to red Vegetative growth and pink surface Reverse aerial growth; by an extremely course aerial mycelium;

and by a sparse growth of monoverticiliate or brvertrcfl- Bennews agar Pink aerialgmwth Rem late sporophores. These morphological characteristics Q a izeks sucr ose agar. g ge pip er a gro t h p1nk together with the cultural characteristics given below 385; ,3 gif i f 2 ti fi fitg pgg distinguish the organism as a new species. g i igg r- F ir pink aerial growth. lginik-red.

. 21511 BICHBI 0 e. In Table I, Sti'eptomyces fervens is characterized 210- g cofdlng its ability to utilize cafbofl Compounds by Further cultural characteristics of S. fervens are the Procfidure 0f Pl'ldham and Gofllleb k 561 shown by its growth on fourteen dIfierent standard cul- 107-l14 (1948)] with the modifications noted in the ture media at selected temperatures as shown in the printed German patent application No. 1,025,893. following table:

TABLE 111 Cultural characteristics of Streptomyces fervens (14 days incubation at 28 C.)

Medium Surface Reverse Other Peptone iron agar No aerial growth. grey vegetative growth Brown His darkening.

9.1% tyrosine agar. Deep pink aerial growth" Pink Fair-brown pigment.

Litmus milk Ring at surfacebrown on top. cream pink on bot- Pink-tan growth at base, slight tan pigment,

tom. Trace pink aerial growth. nitrates reduced.

Synthetic nitrate broth Pink surface pellicle, trace pink aerial growth. 0.

Organic nitrate broth Pink vegetative growth, ring at surface, no aerial Flocculent growth at base, tan pigment, nitrates P1 I f (127) growth. Breduced. t yu fi d 8111. go a 1.11 lOW'Il pigmen que 9 Calcium malate agar Trace pink aerial grow Pink No pigment. 2

Glucose asparagine agar Firm pink aerial growth d0 Pale yellow pigment.

Skim milk agar No aerial growth, pink vegetative growth Pink-tan. N o pigment, no hydrolysis.

Casein starch agar Pink aerial growth in Slight tau, pigment, starch hydrolyzed.

llgutrietnt starch agar Slight pink aerial growth Red-pink--. Tan pigment, starch hydrolyzed.

enne ts agar a 18, 24, and 28 0...... Compact pink aerial growth Redorange Slight-tan pigment. 37 0 Trace maroon aerial growth Maro Do.

Fair pink-white aerial growth Pinlcwhite- No pigment. Fair pink aerialgrowth Pink Do.

Compact pink aerial growth Red-orange. Slight-tan pigment. Peach-pink aerial growth Red-brown- Do. Trace pink aerial grow h Maroon Do.

Fervenulin is produced when the particular strain of S. fervens characterized above is grown in an aqueous nutrient medium under submerged aerobic conditions, and preferably, in a nutrient medium containing a carbon source, such as an assimilable carbohydrate, and a nitrogen source, such'as an assimilable nitrogen compound or a proteinaceous material. It is to be understood, however, that the invention also embraces other fervenulinproducing strains of S. fervens, such strains being readily produced and isolated by routinely applied isolation and strain modification methods which include selection of cultured organisms and exposure of these organisms to modifying means such as X-ray, ultraviolet light, chemical agents such as nitrogen mustards, and the like. It is to be understood also that for the preparation of limited amounts, shaken flasks and surface cultures in bottles can be employed. The presently preferred sources of carbohydrates include glucose, dextrose, sucrose, dextrin,'molasses (black strap and high test or invert), corn meal (refined and unrefined), and starch (bolted and soluble), and combinations thereof. Other suitable carbon sources are maltose, lactose, galactose, glycerol, mannitol, soy,- bean oil, animal and vegetableoils, and the like. Preferred sources of nitrogen include protein sources such as cotton seed meal, soybean meal, soya flour, fish meal, peanut meal, defatted soybean meal, peptone (meat, soya, egg, milk), and the like. Other suitable sources are milk proteins, brewers yeast (dried yeast cells obtained from a beer fermentation) or yeas'textra'ct, corn gluten meal, corn steep liquor, fish stick liquor, animal stick liquor, distillers solubles, trypticase, tryptone, beef extract, N-Z amine A (an enzymatic hydrolysate of casein), N-Z amine B (a pancreatiodi est of casein), N-Z amine E (a tryptic digest of casein), proteolyzed milk, meat, soya and egg products, amino acid mixtures, inorganic nitrogen as nitrate and ammonium salts and the like. Combinations' of two or more of these nitrogen sources can be used advantageously.

'Nutrient inorganic salts, for example, salts capable of yielding ions such as potassium, sodium, calcium, phosphate, chloride, sulfate, and the like, can be incorporated advantageously in the medium. Essential trace elements such as zinc, magnesium, manganese, cobalt, iron, and the like, can also be included in the culture medium for growing S. fervens. Such trace elements are commonly.

where cobalamines (vitamin B and vitamin B -like products) are desired, and these by-products then recovered by conventional methods. Similarly, steroid precursors, such as progesterone or Reichsteins compounds or S acetate, can be added to obtain an oxidized steroid.

Suitable temperatures for the growth of S. fervens and for the production of fervenulin lie between about 18-37" C. The time required under suitable submerged aerobic fermentation conditions for optimum production of fervenulin is generally from 2-10 days. The culture medium, prior to inoculation With the microorganism, advantageously is adjusted to a pH between about 7.0 and about 8.0, and preferably to about pH 7.5. Themedium normally stays alkalineduring fermentation, the final pH being dependent, in part, on the initial pH of the culture medium, the buffers present, and the like.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetable form of the microorganism for inoculation to avoid a pronounced lag in the production of fervenulin and the attendant inefiicient utilization of the equipment. Accordingly, it is desirable first'to produce a maltose tryptone agar slant which is inoculated with the material from a ly ophilized culture or with an aliquot of a soil culture A vegetative inoculum of the microorganism is then produced by inoculating a relatively small amount of culture medium with material scraped from the agar slant. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum as produced can be the same as, or different from, that utilized for the produc: tion of fervenulin as long as it is such that a good growth of the organism is obtained.

The rate of production of fervenulin and the concentration of fervenulin in the culture medium are readily followed during the growth period of the microorganism by testing samples of the culture medium for antibacterial activity against an organism known to be susceptible to fervenulin e.g., Klebsiella pneumoniae, by standard agar diffusion or turbidimetric test procedures, by fractionating a sample by chromatography and measuring the zones located by a Cary spectrophotometer by their absorption at 239 m or by extraction into methylene chloride at pH 6 followed by absorbance measurement at 342 mu.

Fervenulin can be removed from the culture medium by adsorptive techniques including adsorption on carbon or like capillary adsorbent, and .elution therefrom with a suitable eluting agent 'or by solvent extraction procedures.

According to a preferred procedure the Whole beer is filtered, the fervenulin is extracted from the filtrate with a water-immiscible organic solvent such as a lower haloalkane like methylene chloride or chloroform, a lower alkyl alkanoate' like ethyl or butyl acetate, a lower alkanone like butanone, or pentanone (either isomer), or a lower alkanol such as butanol (except t'-butanol) or pentanol (all isomers). The fervenulin can be crystallized directly from the solvent extract by evaporation of the solvent, addition of a hydrocarbon solvent such as hexane, or evaporation of the solvent (to an oil or to dryness) plus crystallization from another solvent such as acetone, methanol, ethanol, and the like. Crude fervenulin, obtained by crystallization as above or simply by evaporation of the extraction solvent and an oil, can be further purified by countercurrent extraction, countercurrent distribution, or by partition chromatography. Fervenulin can be recrystallized from any of the solvents mentioned above particularly acetone, ethyl acetate, methylene chloride, methanol, ethanol, and preferably chloroform.

7 The whole beer can be filtered at a pH between 3 and 9,

and preferably between pH 7-8. Ordinarily it will not be necessary to adjust the pH before filtering as the pH at the end of the fermentation will ordinarily be between pH 7 and pH 8. Extraction of the filtrate is advantageously effected at a pH between 3 and 7, preferably at about pH 6, because fervenulin though a neutral compound, tends to become degraded in alkaline solutions. The purification of crystalline fervenulin can also be accomplished by sublimation at about C. and 10 microns pressure.

Fervenulin exhibits a characteristic pattern given below with bioautographed papergrams. Location of zones is by bioautography with Klebsiella pneumoniae. The composition of the solvent systems (the remainder being water) and R; values are as follows:

Fervenulin has a broad antimicrobial spectrum as exhibited by its activity against bacteria such as: Klebsiella pneumoniae, Escherichia coli, Aerobacter aerogenes,. Salmonella paratyphi B, Salmonella typhosa, Salmonella pullorum, Salmonella schottmuelleri, Pasteurella maltop-toluene sulfonic acid;

'cida, Proteus vulgaris, Diplococcuspr l eumoniac, Streptococcus hemolytz'cus, Streptococcus viridans, Micrococ basis) of fervenulin. The fervenulin present was then harvested as follows: I

The whole beer was adjusted to pH 8.5 and filtered. The filtrate was adjusted to pH 6 and extracted with as: Agrobacter tumefaciens, Corynebacterium faciens, 5 methylene chloride, 85 liters being used. The methylene Erwinia amylovora, Corynebacterium michiganensis, chloride extract was concentrated to an oilby evapora- Xanthomonas campestris, Xanthomonas malvacearum, tion of the solvent. A sample, 20 grams, of the oil was Xanthomonas pelargoni, and Xanthomonas vesicatoria; dried on a porous plate to a solid residue. A sample, against fungi such as: Hiszoplasma capsulatum, Blasto- 1.5 grams, of the solid residue was dissolved in 100 ml. myces dermatitidis, Cryptococcus neoformans, Micro- 10 of chloroform and stirred with 1.5 grams of activated sporum canis, Trichophyton interdigitale, Nocardia carbon for 45 minutes. The mixture was filtered and the asteroides, Hormodendi'um compactum, Phialophora chloroform was removed by evaporation to yield 394 verrucosa, Monosp rium apiospermum, Sporotrichum mg. of yellow crystalline fervenulin, Preparation 1, which schenckii, Coccidioz'a'es immitis, Geotrichum sp., and assayed 740 meg/mg. of fervenulin. Microsporum andouini; and against protozoa such as Preparahon 1 exhibited the following antibacterial Trichomonas foetus, Trichomonas vaginalis, and Entaspectrum demonstratmg its activity against gram positive moeba histolytica. and gram negative bacteria and mycobacteria:

The following examples are illustrative of the process and products of the present invention, but are not to llrle vgnjmalinv i itory conconstrued as lim ting. All percentag s are by welg t Testorganisms centrafion and all solvent mixture proportions are by volume unless (MIC) otherwise noted. (meg/ml) EXAMPLE 1 Ba illus sublilis 5 M' -IIIIIIIII A lyophrlrzed culture of Streptomycesfervens, NR RL igggzf igfgggg 3 2755, was used to seed the following sterile agar medium gi pi occus girid ps 1 .0 on tubed slants: 3 1 pnez. 100.0 G Pasteurella 'mwlfmiflfl 3 1 f3 1 as rdeus uglms l g altose 5 Escherichia col 50,0 ryptone 5 fiseugwgomas aeru inosa 100.0 em as er aer0genes 25.0 K2HPO4 o Salmonella schottmuelle 0 5 NaCl Sul slla puratyphi B 50.0 FeSO 0.1 Klebsz'ella m. 25. 0 4 0 lLfi cobacterium tuberculo is bovis 50.0 Agar 2 Salmonella typhosa 50.0 Deiomzed water to make 1 hter. 58-8 The slants were incubated for 7 days at 30 C., after I which time sporulafion was complete The Spores from the fo.1owmg spectrum against plant pathogenic bacteria: the agar slants were used, in an aqueous suspension, to inoculate 100 milliliters of preseed medium in a 500 40 Test organisms MIC milliliter Erlenmeyer flask. The sterile preseed medium (mew/m J conslsted Ag10bacter[umcfaciens 200 Grams gorynebaclerz'um facz'en 100 nuim'a' "a" 200 Cotton seed meal Corymbacterium michiganensim 100 Glucose 40 4:5 Xanthomonas campestris 200 T p w r to m 1 azaleas else??? Adjusted to pH 7.2 before sterilizing. Xlmthomonus vesicatorz'a 200 The seed flask was incubated for 72 hours at 28 C.

the 0 W1 0 nt v on a reciprocating shaker after which it was used as an i no a mmual Spectrum inoculum for a 20 liter seed fermentor in the amount of n approximately 3%. The 20 liter seed fermentor con- Test orgamsms Qg E tained a sterile medium consisting of:

Grams N ocardia astemz'des 1,000. 0 Glucose 20 1.3.8818 Kay-soy 1 10 geotritkhuk'n sp 1, 000. 0 ormo en rum compactum 1,000.0 lewel yeast Q Cryptococcus neufor1nans 100. (l Ammonium ChlOnGe 5 SHzsZopla-sma cugmulglum. 0(110.0 2 porrichum sc e-nc tin--. 1, 0.0 (3319mm carbfmate 4 M'onasporz'um a'piospermum 1,000.0 Sodium chloride 3 Microsporum J 100.0 Tap water to make 1 liter g;,'; ;,9;g;g*? a l 27L 8 1g 1 Fat extracted soybean meal, finely milled. Phmzflphom vermcom 1,0001) {The calcium carbonate was added after the medium was ad usted to pH 7 .2 and before sterilization. 1 1001] partial inhibition The 20 liter seed fermentor was incubated for 4? hours and the following antiprotozoal spectrum: at 28 C. and aerated at the rate of 6 standard hters or about 0.2 standard cubic foot (s.c.f.) of air per minute Test organism M10 and agitated with a sweep stirrer. The 20 hter seed es/ 'fermentor was used to inoculate 250 liters of the same medium in a 100 gallon fermentation tank. 900 milligggggggggf m; liters of lard oil were added during the fermentation to control foaming. The tank was agitated with a propeller D and aerated at the rate of standard liters (about 2.6 EXAM LE 2 s.c.f.) of air per minute. After 66 hours of fermentation The slant and preseed were prepared and used followthe beer assayed 134 meg/ml. (45 mcgJmg. on a dry 75 mg the procedure of Example 1'. The preseed was used at the rate of 2% to inoculate 300 liters of seed medium (following the procedure of Example. 1) in a fermentc-r tank. The seed tank was incubated for 60 hours at 28 C., agitated with a propeller, and aerated at the rate of 8 s.c.f. of air per minute. The seed was used to inoculate 5000 liters of the same culture medium prepared in a 2000 gallon fermentation tank. The fermentor was incubated at 28 C., agitated by a propeller at 166 r.p.m., and aerated at the rate of 80 s.c.f. of air per minute. The fervenulin activity (the whole beer assayed 120 meg/ml. of fervenulin) in the whole beerwas recovered after 42 hours of fermentation as follows:

The whole beer was adjusted to pH 8 and filtered.

The clear filtrate was adjusted to pH 5.9 and extracted with methylene chloride. The methylenechloride ex- ,tract was concentrated to an oil which was leached with upper phase of a solvent system consisting of acetone:nhexanezwater in the volume proportions :3:1, respectively. The leached extract was evaporated to yield crude fervenulin as a dark brown semicrystalline material, A sample, 815 grams, of this semicrystalline crude fervenulin was leached 6 timm with a total of 400 milliliters of acetone, at Dry Ice temperature. The leached crystals were dissolved in 400 milliliters of solvent-systern consisting of ethyl acetatezacetone in the volume proportions 3:1, respectively, and stirred for 5 minutes with 3 grams of activated carbon. The mixture was filtered. The filtrate, a yellow solution, was evaporated under a stream of nitrogen to yield 6 grams of yellow crystalline fervenulin, Preparation 2a, which assayed 880 mcg./ mg. of fervenulin. Preparation 2a was recrystallized from 150 ml. of acetone and 50 ml. of water to yield 3.5 grams of crystalline fervenulin, Preparation 2b, a brilliant yel-' low crystalline material which melted at 178179 C.,

Preparation 2b gave negative ninhydrin, ferric chloride, Tollens,

and assayed 1000 meg/mg. of fervenulin.

about 2 mg./ml., in hot water to about 40 mg./ml.; and

The material, Preparawas insoluble in hydrocarbons. tion 2b was stable to acid hydrolysis and was recovered intact aftertreatment with 6 N HCl on a steam bath for 40 hours.

EXAMPLE 3 A sample, 980 mg, of a material similar to Preparation 2a which assayed 900 meg/mg. of fervenulin, was further purified by counter-current distribution. using the solvent system benezenezmethanolz water in the volume proportions l:1:0.2, respectively, through 216 transfers.

j A single color band was observed between tubes 80-115.

The single weight peak occured in tube 99. The material,- in tubes 90l()3, was pooled and then evaporated .to a dry crystalline residue. The crystalline residue was recrystallized from acetone and water to yield 450 mgs. of

purse crystalline fervenulin, Preparation. 32, melting at 176-178 C., assaying 1000 mcg/mgof fervenulin and having the following elemental analysis:

Calculated for C7H7N5O2: C, 43.52; H, 3.65; N, 36.26;

'0 16.56." Found: C, 43.83; H, 3.73; N, 35.99; 0, 17.27.

This analysis coupled with the molecular weight determination given in the following table indicates a molecular formula of C H N O Method Compound Calculated Found X-Ray A -2 C7H7N502... 193. 18 189 Vapor oressur r 5 5 193.18 190 Saponillcation- C H N O2m 193. 18 18G The pure crystalline fervenulin is further characterized as follows: 1

The infrared spectrum, FIGURE 1, shows acornplete absence of any absorption in the OH/NH region and exhibits characteristic absorption bandsexpressed in reciprocal centimeters at the following frequencies: 3040,v 1718, 1675,1575, 1535, 1495, 1435, 1415, 1397, 1296,. 1255, 1219, 1115, 1085, 1044, 995, 962, 930, 884, 815, 740, 730, 709.

The ultraviolet spectrum, FIGURE 2, exhibits a maximum absorption at 239 my. where (coefiicient'of extinction) a=87, a small maximum between 270-280 mu where a=8.5-9.0 and a maximum at 340 my. where Crystallization from chloroform gives bright yellow crystals having the following properties:

Melting point a 178-179 C. Crystal system orthorhombic. Crystal class Rhariibic dipyramia Crystal habit Lamellar. Axial ratio a:b:c,0.883:0.316. Unit cell dimensions a= 15.86 A. [7:17.96 V A., 0:5.667 A. Crystal density 1.555. Formula weights per cell 8. Molecular weight 189. Optic sign Negative. Dispersion Extreme, V R. Optic axial angle 2V=23 30 (calc.). Optic orientation b=X, c =Y, a=Z. Refractive indices' (for sodium a=1.584, 5:1.706, 7:1.712.

Crystalline fervenulin also exhibits large negative birefringence, and sublimes readily at 70 C. and 10 microns pressure.

It is to be understood that the invention is not to be limited to the exact details of operation or exact compounds shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the scope of the appended claims.

7 We claim:.

1. A novel composition of matter assaying at least 4.5

meg/mg. of the compound, fervenulin, which in its es- 7 sentially pure crystalline form is characterized by a yellow orthorhombic crystalline form; by a melting point of 178-179 C.; by a molecular formula of C H N O by solubility in methanol, ethanol, acetone, chloroform, ethyl acetate and water; 'by .insolubility in hydrocarbon solvents; and by exhibiting characteristic infrared and ultraviolet absorption spectra as shown in the drawings, FIGURES 1 and 2, respectively.

2. Crystalline fervenulin, as defined in claim 1.

. 3. A novel composition of matter consisting essentially of fervenulin as defined in claim 1.

4. The process which comprises cultivating Streptomycesjervens in an aqueous nutrient medium under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium. a

5. The process which comprises cultivating Streptomyces fervens in an aqueous nutrient medium under aerobic conditions until substantial activity is imparted to said medium by production of fervenulin and isolating fervenulin from the culture medium, said fervenulin in its essentially pure crystalline form being characterized as in claim 1.

6. A process which comprises cultivating under aerobic conditions Strepromyces fervens in an aqueous nutrient medium at a temperature of about 18 C. to about 37 C. for a period between about 2 and about 10 days until substantial activity is imparted to said medium by production of fervenulin, separating the fervenulin from the culture medium and isolating fervenulin therefrom, said fervenulin in its essentially pure crystalline form being characterized as in claim 1.

7. The process of claim 5 which comprises isolating fervenulin from the culture medium by extraction with a water-immiscible solvent for fervenulin.

8. The process of claim 7 which comprises isolating fervenulin from the culture medium by extraction with a water-immiscible organic solvent for fervenulin selected from the group consisting of halogenated hydrocarbons,

lower alkanols, lower alkanones, and lower alkyl esters. 1

9. The process of claim 8 wherein the halogenated hydrocarbon is methylene chloride.

10 References Cited in the file of this patent UNITED STATES PATENTS Haines et a1. Aug. 18, 1953 OTHER REFERENCES Pridham et 211.: Applied Microbiology, 1958, pp. 52-79, vol. 6.

De Boer et al.: Antibiotics Annual 1959-1960, pages 220-226; pub. 1960; pub. by Antibiotics, Inc. N.Y.C.

Eble et al.: Antibiotics Annual 1959-1960, pages 227- 229; pub. 1960; pub. by Antibiotics, Inc., N.Y.C.

J. of Bacteriology, October 1956, page 4.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No, 3 O22,22O February 20 1962 Clarence De Boer et a1.

It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 2, TABLE I, opposite "l4." forflnsulin" read Inulin column 5, line 75, for "45 mcg./mg.," read 4.5 mega/mg. column 7 line 61 for "purse" read pure column 8 line 22, for a:b:c: 00883201516" read a:b:c, O..883:l:Oq316 Signed and sealed this 19th day of June 1962,

(SEAL) Attest:

ERNEST w. SWIDER DAVID L LADD Attesting Officer Commissioner of Patents 

1. A NOVEL COMPOSITION OF MATTER ASSAYING AT LEAST 4.5 MCG./MG. OF THE COMPOUND, FERVENULIN, WHICH IN ITS ESSENTIALLY PURE CRYSTALLINE FORM IS CHARACTERIZED BY A YELLOW ORTHORHOMBIC CRYSTALLINE FORM; BY A MELTING POINT OF 178-179*C.; BY A MOLECULAR FORMULA OF C7H7H5O2; BY SOLUBILITY IN METHANOL, ETHANOL, ACETONE, CHLOROFORM, ETHYL ACETATE AND WATER; BY INSOLUBILITY IN HYDROCARBON 